The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

Mitochondria are organelles derived from an intracellular alpha-proteobacterium. The biogenesis of mitochondria relies on the assembly of beta-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an alpha-proteobacterium, and the BAM (beta-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A approximately 150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA(His) have distinctive identity elements, we constructed E. coli tRNA(His) CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA(His) CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA(His) CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA(His) CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.

To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the hfq deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid-associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA-binding activity is intimately linked to the action of DNA replication.

Cell polarization is an integral part of many unrelated bacterial processes. How intrinsic cell polarization is achieved is poorly understood. Here, we provide evidence that Caulobacter crescentus uses a multimeric pole-organizing factor (PopZ) that serves as a hub to concurrently achieve several polarizing functions. During chromosome segregation, polar PopZ captures the ParB*ori complex and thereby anchors sister chromosomes at opposite poles. This step is essential for stabilizing bipolar gradients of a cell division inhibitor and setting up division near midcell. PopZ also affects polar stalk morphogenesis and mediates the polar localization of the morphogenetic and cell cycle signaling proteins CckA and DivJ. Polar accumulation of PopZ, which is central to its polarizing activity, can be achieved independently of division and does not appear to be dictated by the pole curvature. Instead, evidence suggests that localization of PopZ largely relies on PopZ multimerization in chromosome-free regions, consistent with a self-organizing mechanism.

In bacteria, ParABS systems mediate intracellular transport of various cargos, including chromosomal regions in Caulobacter crescentus. Transport of the ParB/parS partition complex requires the DNA-binding activity of ParA, which transiently tethers the partition complex during translocation. In C. crescentus, the directionality of the transport is set up by a gradient of ParA whose concentration gradually increases from one end of the cell (old pole) to the other (new pole). Importantly, this ParA gradient is already observed before DNA replication and segregation are initiated when the partition complex is anchored at the old pole. How such micron-scale ParA pattern is established and maintained before the initiation of chromosome segregation has not been experimentally established. Although the stimulation of ParA ATPase activity by the localized ParB/parS partition complex is thought to be involved, this activity alone cannot quantitatively describe the ParA pattern observed inside cells. Instead, our experimental and theoretical study shows that the missing key component for achieving the experimentally observed steady-state ParA patterning is the slow mobility of ParA dimers (D ∼10(-3)μm(2)/s) due to intermittent DNA binding. Our model recapitulates the entire steady-state ParA distribution observed experimentally, including the shape of the gradient as well as ParA accumulation at the location of the partition complex. Stochastic simulations suggest that cell-to-cell variability in ParA pattern is due to the low ParA copy number in C. crescentus cells. The model also accounts for an apparent exclusion of ParA from regions with small spacing between partition complexes observed in filamentous cells. Collectively, our work demonstrates that in addition to its function in mediating transport, the conserved DNA-binding property of ParA has a critical function before DNA segregation by setting up a ParA pattern required for transport directionality.

The genetic network involved in the bacterial cell cycle is poorly understood even though it underpins the remarkable ability of bacteria to proliferate. How such network evolves is even less clear. The major aims of this work were to identify and examine the genes and pathways that are differentially expressed during the Caulobacter crescentus cell cycle, and to analyze the evolutionary features of the cell cycle network.

The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation.

Cell size control is an intrinsic feature of the cell cycle. In bacteria, cell growth and division are thought to be coupled through a cell size threshold. Here, we provide direct experimental evidence disproving the critical size paradigm. Instead, we show through single-cell microscopy and modeling that the evolutionarily distant bacteria Escherichia coli and Caulobacter crescentus achieve cell size homeostasis by growing, on average, the same amount between divisions, irrespective of cell length at birth. This simple mechanism provides a remarkably robust cell size control without the need of being precise, abating size deviations exponentially within a few generations. This size homeostasis mechanism is broadly applicable for symmetric and asymmetric divisions, as well as for different growth rates. Furthermore, our data suggest that constant size extension is implemented at or close to division. Altogether, our findings provide fundamentally distinct governing principles for cell size and cell-cycle control in bacteria.

Polarity is often an intrinsic property of the cell, yet little is known about its origin or its maintenance over generations. Here we identify a landmark protein, TipN, which acts as a spatial and temporal cue for setting up the correct polarity in the bacterium Caulobacter crescentus. TipN marks the new pole throughout most of the cell cycle, and its relocation to the nascent poles at the end of division provides a preexisting reference point for orienting the polarity axis in the progeny. Deletion of tipN causes pleiotropic polarity defects, including frequently reversed asymmetry in progeny size and mislocalization of proteins and organelles. Ectopic localization of TipN along the lateral side of the cell creates new axes of polarity leading to cell branching and formation of competent cell poles. Localization defects of the actin-like protein MreB in the DeltatipN mutant suggest that TipN is upstream of MreB in regulating cell polarity.

The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria.DOI: http://dx.doi.org/10.7554/eLife.02758.001.

Bacterial cell poles constitute defined subcellular domains where numerous proteins localize, often at specific times, to affect various physiological processes. How pole recognition occurs and what governs the timing of protein localization are often unknown. In this paper, we investigate the mechanisms governing the localization of PopZ, a chromosome-anchoring protein whose unipolar to bipolar localization pattern is critical for cell cycle progression in Caulobacter crescentus. We provide evidence that polar localization of PopZ relied on its self-assembly into a higher-order structure (matrix) and that the unipolar to bipolar transition was coupled to the asymmetric distribution of ParA during the translocation of the origin-proximal ParB-parS partition complex. Collectively, our data suggest a model in which a local increase of ParA concentration promotes the assembly of a PopZ matrix precisely when and where this matrix is needed. Such coupling of protein assembly with a cell cycle-associated molecular asymmetry may represent a principle of cellular organization for controlling protein localization in both time and space.

The β-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, β-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli.

Crescentin is a bacterial filament-forming protein that exhibits domain organization features found in metazoan intermediate filament (IF) proteins. Structure-function studies of eukaryotic IFs have been hindered by a lack of simple genetic systems and easily quantifiable phenotypes. Here we exploit the characteristic localization of the crescentin structure along the inner curvature of Caulobacter crescentus cells and the loss of cell curvature associated with impaired crescentin function to analyze the importance of the domain organization of crescentin. By combining biochemistry and ultrastructural analysis in vitro with cellular localization and functional studies, we show that crescentin requires its distinctive domain organization, and furthermore that different structural elements have distinct structural and functional contributions. The head domain can be functionally subdivided into two subdomains; the first (amino-terminal) is required for function but not assembly, while the second is necessary for structure assembly. The rod domain is similarly required for structure assembly, and the linker L1 appears important to prevent runaway assembly into nonfunctional aggregates. The data also suggest that the stutter and the tail domain have critical functional roles in stabilizing crescentin structures against disassembly by monovalent cations in the cytoplasm. This study suggests that the IF-like behavior of crescentin is a consequence of its domain organization, implying that the IF protein layout is an adaptable cytoskeletal motif, much like the actin and tubulin folds, that is broadly exploited for various functions throughout life from bacteria to humans.